Of central importance to thrombolysis is the activation of plasminogen (Plg) to form plasmin (Pm) which catalyze the degradation of fibrin clots. Streptokinase (SK), a bacterial protein, is a plasminogen activator widely used in the clinical treatment of clotting disorders, including myocardial infarction. Unlike tissue plasminogen activator and urokinase, SK is not a protease. SK and Plg form a non-covalent complex, which is proteolytically active and converts other Plg molecules to Pm leading to fibrinolysis. The detailed activation mechanism of Plg by SK remains unclear due largely to the lack of information on their three-dimensional structure. Since both proteins contain multiple domains which may be flexibly connected, the crystallization of the native proteins for structural studies has been unsuccessful so far. Using protein engineering the applicants have produced a functional core of the SK-Plg complex. An active-site mutant of a truncated Plg (muPlg*) complexed with SK has produced crystals which diffract to high resolution. The current application proposes three aims. First, the crystal structure of the SK-muPlg* complex on hand will be determined at high resolution. Second, the understanding of the SK and Plg interaction from the core complex structure will be extended to the full size proteins. This will be accomplished by studying the interaction and structure of the domain of the proteins. The third aim is to gain an understanding of how SK-binding activates Plg. This will be done by comparing the structures of the proteins in both free state and in complexes and by designed mutagenesis. The structural and functional understanding of the SK-Plg interaction may promote design of better thrombolytic agents for clinical treatment.